(a) Field of the Invention
The present invention relates to inhibitors of the eukaryotic ribosome recruitment phase of translation initiation and their use as antiproliferative and/or chemotherapeutic agents and/or as adjuvants in combination therapy.
(b) Description of Prior Art
The ribosome recruitment step of translation initiation is rate-limiting and an important regulatory point whereby cellular environmental cues (e.g.—amino acid starvation, mitogenic stimulation, and hypoxia) are linked to the process of translation (18). Two distinct pathways exist for recruitment of the ribosome to the mRNA template. One mechanism is cap-dependent and is facilitated by the presence of the 5′ cap structure (m7GpppN, wherein N is any nucleotide) on the mRNA. It is catalyzed by the eIF4 class of translation initiation factors and involves the recruitment of ribosomes near the 5′ end of the mRNA template (18). The second mode involves ribosome recruitment in a cap-independent fashion to an internal ribosome entry site (IRES). Initiation factor requirement for internal ribosome binding varies among IRESes, with some not requiring any factors (56).
Translation initiation is the rate-limiting step of protein synthesis. In this complex process, an initiator Met-tRNAi, one molecule of GTP, and a set of initiation factors (eIF1, eIF3, eIF5, eIF1A, and eIF2) assemble on a 40S ribosome to form a 43S pre-initiation complex. Binding of the ribosome to the mRNA is generally the rate-limiting step of the entire process and occurs by one of two mechanisms: a cap-dependent mechanism (FIG. 1) and a cap-independent mechanism involving internal recruitment of the ribosome to the mRNA 5′ UTR. Most translation in eukaryotes is thought to occur by a cap-dependent process involving a set of proteins that are required to recruit the ribosome to the 5′ end of the mRNA template. The key complex here is eIF4F—consisting of 1) eIF4E, the cap-binding protein responsible for binding of the complex to the mRNA cap structure; 2) eIF4A, an RNA helicase required to unwind local secondary structure and thus facilitate access of the ribosome to the mRNA template; and 3) eIF4G, a modular scaffold that mediates mRNA binding of the 43S pre-initiation complex through interactions with eIF3 (which is present on the ribosome). Once bound to the mRNA, the 43S complex is thought to scan the 5′ UTR, supported by ATP hydrolysis, until the appropriate AUG start codon is encountered. Mechanisms of internal initiation, bypassing the need for a 5′ cap structure, have been described and the requirement for the eIF4 class of proteins varies depending on the particular mRNA under study.
Preparation of the mRNA template for cap-dependent ribosome recruitment is mediated by eIF4F, eIF4A, eIF4B, eIF4H, and ATP hydrolysis (18). The eIF4F complex is comprised of three subunits: (i) eIF4E, which binds the mRNA cap structure in an ATP-independent fashion, (ii) eIF4A, an RNA helicase that exhibits RNA-dependent ATPase activity and ATP-stimulated RNA binding activity (15) and, (iii) eIF4G, a modular scaffold that mediates mRNA binding of the 43S pre-initiation complex through interactions with eIF3. eIF4B and eIF4H cooperate with eIF4A to make its helicase activity more processive (44, 45). eIF4A exists as a free form (referred to herein as eIF4Af) and as a subunit of eIF4F (eIF4Ac), and is thought to recycle through the eIF4F complex during initiation (37, 43, 50). It has been previously reported that when localized in the eIF4F complex, eIF4A is approximately 20-fold more active as an RNA helicase compared to its non-complexed state (38, 45). This observation has lead to the proposal that eIF4Ac is the functional helicase for translation initiation (10). The helicase activity of eIF4F is thought to unwind local secondary structure in the 5′ UTR of mRNAs thereby facilitating cap-dependent ribosome recruitment (37, 43, 50). The crystal structure shows that eIF4Af has a distended “dumbbell” structure consisting of two domains (6, 8, 26), which undergo conformational changes in response to RNA and ATP binding (29).
The eIF4A family comprises three members. These are characterized as eIF4AI, eIF4AII, and eIF4AIII. eIF4AI and eIF4AII exhibit 90-95% amino acid homology, are involved in translation, and appear to have similar biological activity in vitro (11, 58). eIF4AIII exhibits in the order of 65% similarity with the other isoforms and is implicated in nonsense-mediated decay (NMD) (9, 13, 35, 48). The foregoing eIF4A isoforms are members of the DEAD-box putative RNA helicase protein family. These, and related DEXD/H (where X is any amino acid) box proteins are characterized by seven highly conserved amino acid sequence motifs implicated in RNA remodeling. These proteins are involved in virtually all aspects of cellular RNA metabolism including ribosome biogenesis, transcription, splicing, translation, and mRNA degradation (for example, see http://www.helicase.net). Targeting compounds, preferably small molecules, to DEXD/H family members would provide mechanistic important insight into the properties of these proteins and help further define their roles in normal and aberrant cellular and developmental processes.
Mammalian Translation Initiation and Cancer
Disruption of one or more steps in the control of protein synthesis has been associated with alterations in the cell cycle and/or regulation of cell growth (18). Thus, proteins involved in translation initiation pathways could act as key regulators of malignant progression. There is compelling evidence supporting the concept that some translation factors are proto-oncogenes (19). Transformed cells generally show higher rates of protein synthesis compared to normal cells (20). Accordingly deregulation of protein synthesis is emerging as a major contributor to cancer progression. Overexpression of certain translation factors can lead to malignant transformation and many of the components of the translation pathways are over-expressed in cancer (19).
Cell Survival and Translation
Translational control is intimately linked to the PI3K/Akt signaling pathway. Activation of this pathway involves the production of a phospholipid, phosphatidylinositol trisphosphate [PIP3], by PI3K (FIG. 2) This in turn triggers a cascade of responses that emanate from Akt activation—ranging from cell growth and proliferation to survival and motility (FIG. 10). One of several downstream targets of Akt is the TSC1 (130 kDa) and TSC2 (200 kDa) protein complex. TSC1 and TSC2 form a heterodimer that appears to be important for the stability of both proteins. TSC2 contains a GAP (GTPase Activating Protein) domain in its C terminus and binding of the TSC1/TSC2 complex to Rheb (a small ras-like GTPase) enhances the GTPase activity of Rheb, converting it to the Rheb-GDP (inactive) form. It has been speculated that Rheb might not require a guanine nucleotide exchange factor (GEF) for its activation as it is present in a high GTP bound state within cells. Subsequent activation of mTOR causes phosphorylation of 4E-BP1, liberating eIF4E from the 4E-BP/eIF4E inhibitory binary complex and stimulating protein synthesis. Complexes between eIF4E and 4E-BP1 and eIF4G have been characterized by high resolution X-ray crystallography and NMR. This pathway is up regulated in a wide range of tumor types (glioblastoma, ovarian, breast, endometrial, hepatocellular carcinoma, melanoma, digestive tract, lung, renal cell carcinoma, and lymphoid) through amplification of the p110 catalytic subunit of PI3K, loss of PTEN phosphatase activity, amplification of Akt, mutations of the tsc1 or tsc2 genes in tuberous sclerosis complex, and overexpression of eIF4E.
Inhibitors of Translation as Chemotherapeutic Agents
Several inhibitors of translation have been tested as anti-cancer agents, the majority of these target the elongation phase of translation. Recent experiments suggest that targeting translation initiation may be a more effective approach.
Compounds that target elongation of translation are discussed below. Sparsomycin and some of its derivatives selectively act on several different human tumors (34). Structure-activity relationship studies indicate that the anti-tumor activity is a consequence of inhibition of protein synthesis (54). Sparsomycin potentiates the cytotoxicity of cisplatin (24) and is selectively active on tumour cells without affecting human bone marrow. Unfortunately, retinotoxicity is one of the major side effects of sparsomycin derivatives in vivo (25). Tenuazonic acid has been tested in several systems for its chemotherapeutic effects. It is active in transformed cell lines (27) as well as a chemopreventive agent, preventing the formation of TPA and DMBA induced skin cancer when topically applied before the carcinogen challenge (5). Bouvardin, which inhibits EFI-dependent binding of aminoacyl-tRNA and EF2-dependent translocation of peptidyl-tRNA (59), has also been tested for its anti-neoplastic potential. It is active against murine leukemia ascites cells (10), and enhances the anti-neoplastic activity of cis-platinum (1). In combination with cis-platinum and vincristine, bouvardin does not show potentiation of activity against advance leukemias (1). Didemnin B, which blocks the translocation step of elongation (3), displays encouraging antineoplastic activity in vitro; it inhibits cell growth in human tumor stem cell assays at concentrations from 1 to 100 nM. Structure activity—studies of didemnin B indicate the same rank order in potency of translation inhibition as its anti-proliferative effects on MCF-7 cells (2). Results from phase II clinical trials suggest that didemnin B has little or no activity against advanced human cancers, possibly due to biotransformation (55). A closely related analogue of didemnin B, dehydrodidemnin 6, is ten times more active than didemnin B against murine leukemia cells (as well as in a number of human tumor xenografts) and does not exhibit the cardiotoxicity of didemnin B. Clinical trials have been undertaken with homoharringtonine, and although negative effects with solid tumors (which may be related to the dosing schedules used) have been reported, encouraging results were reported in patients with acute myeloid leukemia, myelodysplastic syndrome, acute promyelocytic leukemia, and chronic myeloid leukemia (28). Currently, structural derivatives are being generated to improve dose-limiting cardiotoxicity.
The management of Acute Lymphoblastic Leukemia (ALL) with Lasparaginase is a good example of inhibiting protein synthesis as a chemotherapeutic approach. Since transformed haematopoietic cells are often unable to synthesize sufficient asparagine for their own metabolism, they recruit it from serum. Depletion of systemic asparagine pools from the serum by administration of L-asparaginase leads to cell death (31). How asparaginase exerts its specificity on leukemic cells remains to be determined, but decreased asparagine levels are associated with inhibition of protein synthesis. One report suggests that L-asparaginase may affect signaling through FRAPImTOR by preventing phosphorylation of 4EBP1 and S6K, although this latter point needs to be verified in ALL cells.
Small molecule inhibitors which affect the initiation phase of translation and that are currently in clinical trials are derivatives of rapamycin. Tumors which harbor PTEN mutations are more sensitive to killing by rapamycin analogues, including CCI-779, RAD001 and AP23573, than their parental cells (32, 40). The activity of rapamycin is due in part to the inhibition the anti-apoptotic effects and the growth promoting activities of eIF4E. Mimosine is a plant amino acid that inhibits translation initiation through its effect on the p170 subunit of eIF3e and eIF5A (12) (4, 17). It exerts mRNA specific effects, such that the translation of ribonucleotide reductase M2 is inhibited. This is also thought to be responsible for the inhibition of DNA synthesis observed when cells are exposed to mimosine. Mimosine has been shown to induce apoptosis in human pancreatic xenografts (60). The identification of additional inhibitors of translation initiation, as described in the current proposal would provide extremely valuable tools for targeting this pathway in transformed cells. In addition to providing tools important for elucidating the mechanism by which the individual protein factors function, these small molecule inhibitors provide the starting reagents for lead development, in which the pathway of translation initiation is specifically targeted for chemotherapy.
Hippurins are cytotoxic polyoxygenated steroids isolated from the marine organism gorgonian Isis hippuris (52). These compounds have been reported to be cytotoxic to cell growth and the activity of a number of structurally related entities have been tested (14, 52). Analogs bearing a spiroketal ring were found to be more cytotoxic than those without this moiety, with hippuristanol being the most active. Some polyoxygenated steroids have been reported to reverse multidrug resistance (51), but the mechanistic details have remained largely unknown. Other metabolites of hippurins have been reported but have not been evaluated for biological activity (21, 42, 46, 47).
It would be highly desirable to be provided with inhibitors of eukaryotic ribosome recruitment as antiproliferative, chemotherapeutic agents and/or adjuvants.
It would be highly desirable to be provided with compounds that inhibit eIF4A-dependent translation initiation as antiproliferative and/or chemotherapeutic agents and/or adjuvants.